Viral RNA replication, a process fundamental to viral pathogenicity, requires specific recognition of RNA features by proteins. RNA-dependent RNA polymerase (RdRp) is a complex composed of viral and cellular proteins that directs viral RNA synthesis from infecting RNA templates. Many viral RdRp proteins have been sequenced and analyzed. However, a comprehensive mechanism describing RNA synthesis is lacking. Consequently, general knowledge of RdRp is significantly less than that of other RNA and DNA polymerases.
Viruses are obligate parasites which depend upon the infected host for many of the basic processes needed for a successful infection. Because viruses depend on the enzymatic and synthetic functions of the host cell, it is very difficult to treat viral infections without affecting cellular processes. For example, interferon causes significant and widespread changes in the cell and hence leads to a number of side effects, including fever, nausea, and other discomforts. Given that several viral diseases are at pandemic proportions, including influenza, AIDS, and hepatitis, the design of effective virus-specific drugs is increasingly important.
Potential antiviral drugs are usually subjected to an intensive and costly screening program to determine whether the drug can preferentially inhibit a viral process. One such drug is Aziduovir, which is utilized more readily by the reverse transcriptase of the human immunodeficiency virus (HIV) than by host cellular polymerases. Yet other potential antiviral therapies, including antisense molecules and ribozymes, are difficult to produce due to the need for complex molecular recombinant technology.
Thus, there is a continuing need for agents that specifically inhibit viral replication.
The invention provides an isolated and purified nucleic acid sequence that can be used as a specific inhibitor of viral RNA synthesis, i.e., an inhibitor of the viral polymerase. In particular, the present invention provides an isolated and purified oligonucleotide of at least four nucleotides, wherein the oligonucleotide comprises a viral nucleic acid sequence which includes the viral initiation nucleotide. Preferably, the oligonucleotide includes viral initiation sequences and optionally viral promoter sequences.
As described hereinbelow, the sequences needed for the replication of a (+) single strand RNA virus, brome mosaic virus (BMV), have been determined. These sequences are needed to correctly and efficiently initiate RNA synthesis. Mutations in the promoter or initiation sequences of BMV abolish RNA synthesis by the BMV RNA-dependent RNA polymerase (RdRp). Moreover, as the promoter and initiation sequences are conserved in several RNA viruses, the mechanism of RNA synthesis is likely to be very similar in all RNA viruses with genomes of (+)-sense RNA. Furthermore, mutations in the initiation sequences will not result in a decrease in the efficacy of the nucleic acid sequences of the invention, as these mutations result in a decrease in viral replication.
The oligonucleotide preferably includes the initiation nucleotide and at least the first, and more preferably the first two nucleotides which are 3xe2x80x2 of the initiation nucleotide in the viral sequence. An oligonucleotide of the invention, or a plurality thereof, is useful to prepare an antiviral composition. The antiviral compositions of the invention are particularly useful in methods to inhibit the infection or replication of (+) single strand RNA viruses, such as the alpha virus superfamily, which includes bacterial, plant and animal viruses. The oligonucleotide of the invention may comprise DNA or RNA, or a hybrid thereof. Preferably, the oligonucleotide is DNA. It is also preferred that the oligonucleotide of the invention comprises at least about four to about fifty, more preferably at least about eight to about forty, even more preferably at least about twenty to about thirty-five, and yet even more preferably at least about eight to about thirteen, contiguous nucleotide bases that preferably have at least about 90%, more preferably at least about 95%, and even more preferably 100%, contiguous nucleotide sequence identity to a particular wild-type viral sequence. However, the invention is not limited to oligonucleotides having identity to a wild type viral promoter and/or initiation sequence, as nucleotides may be substituted with analogs thereof, e.g., analogs of nucleotide bases. Preferred nucleotide analogs are those which have enhanced adsorption for human cells and/or enhanced stability in human serum relative to native nucleotides. For example, the incorporation of sulfur residues in an oligonucleotide, e.g., in the RNA backbone, increases the binding of RdRp to the oligonucleotide. Oligonucleotides may be prepared by methods well known to the art.
Also provided is a method to inhibit or treat a viral infection which employs at least one oligonucleotide of the invention. The method comprises the administration to a cell having, or suspected of having, a viral infection, an effective amount of at least one oligonucleotide of the invention, wherein the oligonucleotide comprises the initiation nucleotide for the virus. For example, as shown in FIG. 7, a DNA oligonucleotide of the invention inhibits viral RNA replication in vitro at a concentration in the micromolar range. The DNA oligonucleotides of the invention may be modified in such a manner so as to prevent RdRp release, e.g., by the incorporation of abasic residues, bulky blocking groups, terminal nucleotides with aliphatic moieties, or covalent crosslinker.
The present invention may be superior to other known antiviral agents because it requires only the nucleic acid sequence(s) for initiation of viral nucleic acid synthesis. Thus, the sequence is readily prepared, does not require complex manipulation by molecular biology techniques, and only a minimal screening regime is necessary. Moreover, the present oligonucleotides may be modified to increase their efficacy at lower concentrations, e.g., RNA oligonucleotides may be modified so as to contain nucleotide analogs or be subjected to circularization, to provide agents that are more stable in vivo so as to, for example, have increased resistance to degradation in serum. Given that every virus will have a minimum of one, and more likely at least two, sequences needed for the initiation of nucleic acid synthesis, at least one, preferably two, and more preferably three (for viruses with subgenomic promoters), therapeutics can be designed for every virus. In particular, a nucleic acid sequence can be prepared to inhibit genomic as well as subgenomic (internal initiation from subgenomic promoters) nucleic acid synthesis. In addition, a particular inhibitor may also be used to disrupt other steps in viral replication, e.g., translation, protein and nucleic acid modification, which may be tightly linked to the initiation of RNA synthesis. Thus, an oligonucleotide of the invention may be employed in combination with other anti-viral agents.
As used herein, the terms xe2x80x9cisolated and/or purifiedxe2x80x9d refer to in vitro preparation, isolation and/or purification of an oligonucleotide of the invention, so that it is not associated with in vivo substances.